1. Genes that Affect the Assembly of the Nervous System in Drosophila Embryos. Genes that affect the assembly of the developing nervous system were screened by RNA interference. Five thousand eight hundred double-stranded RNA preparations, each corresponding to a different gene, were synthesized and approximately three thousand two hundred and fifty double-stranded RNA preparations were injected into embryos and screened. Twenty seven genes were found whose gene products are required for the development of the central nervous system. These genes include eight protein kinases, two gene regulators, five proteins involved in cell signaling pathways, and five proteins with unknown functions. An RNA interference-based approach to functional genomics in a Drosophila cultured cell system was used with a rapid screening method that was sufficiently sensitive to detect known maternal and zygotic functions in Hedgehog signaling, as well as the functions of two previously unknown Hedgehog pathway components. One gene product is a cell surface protein required for Hedgehog signal reception (dally-like protein) and the other (Casein Kinase Ia) is a candidate tumor suppressor because of its role in regulating basal activities of both Hedgehog and Wingless pathways. 2. vnd/NK-2 Homeobox Gene. The three-dimensional solution structure obtained by NMR of the A35T mutant vnd/NK-2 homeodomain bound to the vnd/NK-2 consensus 16 bp DNA sequence was determined. This mutation to threonine from alanine in position 35 in helix II of the vnd/NK-2 homeodomain is associated with early embryonic lethality in Drosophila melanogaster. Although the unbound mutant protein is not structured, in the DNA-bound state it adopts the three-helix fold characteristic of all known homeodomains, but with alterations relative to the structure of the wild-type analogue. These structural modifications occur, and are accompanied by a 50-fold reduction in the DNA binding affinity, even though most of the protein-DNA interactions originally seen for the wild-type homeodomain are found likewise in the threonine analogue. Alterations include torsional angle changes in the loop between helix I and helix II, and in the turn between helix II and helix III, as well as in a distortion of the usual antiparallel orientation of helix I with respect to helix II. The alteration of the position of leucine 40 in the A35T mutant is proposed to explain the observed 1.27 ppm upfield shift of the corresponding amide proton resonance relative to the value observed for the wild-type analogue. A detailed comparison of the structures of the mutant A35T and wild-type vnd/NK-2 homeodomains bound to the cognate DNA is presented. The consequences of the structural alteration of the DNA-bound A35T mutant vnd/NK-2 protein may constitute the basis of the observed early embryonic lethality.